Fibronectin (FN) was first reported by Morrison et al. [Morrison, P. R. et al., J. Am. Chem. Soc., 70, 3103 (1948)] as one of plasma proteins in 1948. Being a group of multifunctional proteins broadly distributed in various tissues and body fluids, this substance is known to be involved, as a cell adhesion factor, in a large variety of biological events such as the migration, differentiation, proliferation and canceration of cells [Sekiguchi, K.: Cell Engineering, 4, 485-497 (1985)].
Meanwhile, FN synthesized in the liver and existing in the blood is known as plasma FN (pFN), and FN detected on the cultured cell surface and culture supernatant is called cellular FN (cFN) [Sekiguchi et al., J. Biol. Chem., 260 (8), 5105-5114 (1985)]. It has been shown that these species of FN are subject to molecular diversity due to alternative splicing of the early gene transcription product. As the regions subject to such alternative splicing, there are three regions called EDA, EDB and IIICS, and it is believed that a large number of molecular species occur according to varied combinations of expression of these regions. In pFN, the above-mentioned EDA and EDB regions have not been appreciably expressed.
On the other hand, cFN is an FN in which the level of expression of the EDA region mentioned above ([cf. Kornblihtt, A. R., et al., Nucleic Acids Res., 12, 5853-5868 (1984); Mosher, D. F. (ed.), Fibronectin, pp. 2-9, Academic Press, 1989]; while this region is referred to as "ED" in the former reference and as "EDa" in the latter reference, said region is referred to as "EDA" in the present specification) is high. Hereinafter such FN in which this EDA region has been expressed is referred as "EDA-FN" for short. Peters J. H. et al. conjugated a nonacosapeptide with keyhole limpet hemocyanin (KLH) to prepare an immunogen, constructed an anti-EDA-FN polyclonal antibody, and using the antibody, demonstrated that the blood EDA-FN level is markedly elevated in patients with collagen disease accompanied by vasculitis [Am. Rev. Respir. Dis., 38, 167-174 (1988); J. Lab. Clin. Med., 113 (5), 586-597 (1989)]. However, such a polyclonal antibody as mentioned above can be supplied only in limited amounts and may be contaminated with another antibody differing in specificity. In addition, its antibody titer varies from animal individual to animal individual.
It is reported that pFN varies in kidney diseases and thrombosis [Usui, N. and Ehara, E., Japan. J. Clin. Med., 47, Supplement, 1989, pp. 148-151]. As for the significance of this, however, many points remain unclear. No diagnostic technique has been established as yet based on the assay of pFN. No finding has been obtained with regard to EDA-FN.
Under these circumstances, means have been demanded in the art for promoting investigations into the above-mentioned EDA-FN on the molecular level, for enabling molecular species-specific assay (detection) or purification and, in its turn, for enabling the diagnosis of collagen disease (autoimmune disease), inflammatory diseases accompanied by vasculitis, as in kidney diseases, and thrombosis.
It is an object of the invention to provide means capable of satisfying the above demand. Thus, the present invention intends to provide a monoclonal antibody specifically recognizing EDA and therefore specifically reacting with EDA-FN, provide an EDA-associated peptide, in particular a specific peptide capable of serving as an immunogen for the production of the above-mentioned monoclonal antibody and as a tracer for assaying EDA-FN, and provide a technique capable of assaying the desired EDA-FN or EDA not only in the conventional solid system but also in a liquid system using said antibody and so on.